Chemical analysis of liquids, such as water, milk and biological fluids is often desirable or necessary for health maintenance and diagnostic care. Various compositions and elements to facilitate such analyses are known. Such compositions and elements generally include a reagent composition for determining a substance under analysis, identified as an "analyte" herein. The analyte can be a living organism or a nonliving chemical substance. The reagent composition, upon interaction with the analyte, provides a detectable change (e.g. dye formation).
Recently, much work has been directed to developing compositions and elements which are useful for rapid and highly quantitative diagnostic or clinical analysis of biological fluids such as whole blood, serum, plasma, urine and the like.
For example, for the rapid and effective diagnosis and treatment of infectious diseases, it is desirable to be able to detect the bacteria causing the disease as rapidly as possible. Infections of the urinary tract are among the most common bacterial diseases, second in frequency only to infections of the respiratory tract. In fact, in many hospitals, urinary tract infections are the most common form of nosocomial infections, often following the use of in-dwelling catheters and various surgical procedures. Most urinary tract infections (UTI) result from ascending infection by microorganisms introduced through the urethra and vary in severity from an unsuspected infection to a condition of severe systemic disease. Such infections are usually associated with bacterial counts of 100,000 (10.sup.5) or more organisms per ml of urine, a condition referred to as significant bacteriuria. Under normal conditions, urine is sterile, although contamination from the external genitalia may contribute up to 1,000 (10.sup.3) organisms per ml in properly collected and transported specimens.
Significant bacteriuria may be present in a number of pathological conditions involving microbial invasion of any of the tissues of the urinary tract, or may result from simple bacterial multiplication in the urine without tissue invasion. The infection may involve a single site such as the urethra, prostate, bladder, or kidney, although frequently it involves more than one site. Infection restricted to the urine may present itself as asymptomatic bacteriuria, i.e., a condition which manifests no overt signs or symptoms of infection. Early treatment of this condition can prevent the development of more serious conditions, e.g., pyelonephritis (inflammation of the kidney and the renal pelvis). The rapid detection of bacteria by a reliable method would therefore facilitate an early and specific diagnosis.
Further, in order to insure that a prescribed antibiotic is in fact effective in treating an infection, repeated tests during therapy are required. The need for simple, rapid bacteriuria tests is thus clear. Moreover, in view of the frequent unsuspected asymptomatic occurrences of UTI among children, pregnant women, diabetics and geriatric populations, diagnosis of which may require collection and testing of several specimens, bacteriuria tests must be sufficiently simple and economical to permit routine performance. Again, this illustrates the need for a rapid and inexpensive bacteriuria detection method.
Current laboratory methods based on culturing microorganisms, e.g., the calibrated loop-direct streak method, require significant incubation periods (18-24 hours) before results can be determined. These laboratory methods are also time-consuming to perform and require considerable clinical training and facilities.
Known commercial methods for relatively rapid detection of bacteriuria have serious drawbacks. They are tedious, not completely reliable, require complex reagents or instrumentation, and have limited sensitivity to certain microorganisms and susceptibility to drug or other interferences. Hence, the usefulness of known methods is severely limited.
It is also known that bacterial microorganisms can reduce dyes, resulting in a colorless product (i.e. dye bleach). Alternatively, colorless materials, e.g. tetrazolium salts, can be reduced to form a colored formazan dye, as described in U.S. Pat. No. 3,415,718 (issued Dec. 10, 1968 to Forkman et al) and by Guze et al in Am. J. Med. Sci., Dec., 1963, pp. 691-694. However, the use of formazan dyes for detecting microorganisms has several drawbacks. The formazan dyes generally have low extinction coefficients and therefore cannot be used to detect low levels of microorganisms. The tetrazolium salts have structures that are not readily modified to increase the extinction coefficients of the formazan dyes. Some formazan dyes are insoluble in water and can be toxic to the microorganisms.
U.S. Pat. No. 4,144,306 (issued Mar. 13, 1979 to Figueras) describes a multilayer element for analysis of liquids. This element can include an interactive composition which interacts with an analyte to release a preformed, detectable moiety from an immobile carrier nucleus upon oxidation or reduction. Such release generally requires the presence of a highly alkaline medium (i.e. pH greater than 13). The spectral absorption band of the preformed detectable moiety is the same before and after release. In other words, the detectable species is not shiftable from one spectral absorption band to another. Therefore, the reference teaches the use of radiation-blocking layers in the element to screen out unwanted absorption from unreleased detectable moiety during the assay.
U.S. Pat. Nos. 4,108,850 (issued Aug. 22, 1978 to Fields et al) and 4,139,379 (issued Feb. 13, 1979 to Chasman et al) describe ballasted electron-accepting nucleophilic displacement compounds (called BEND compounds therein) which can release dyes or other photographically useful fragments when reduced in the presence of silver halide, an incorporated reducing agent and an electron transfer agent. However, like the compounds described by Figueras, most of these BEND compounds release the desired moieties only in a high pH (13-14) environment. A few BEND compounds, e.g. those having reduction potentials at about -650 mV (in acetonitrile), will release dye at a lower pH. However, dye release from these compounds at low pH (i.e. less than about 9) is very inefficient, i.e. very slow, and would not provide a rapid clinical chemistry assay. BEND compounds that release dyes only at high pH cannot be used in analytical determinations which are generally carried out at physiological pH (i.e. &lt;9). Highly alkaline conditions are undesirable for clinical analysis, and especially for detection of microorganisms because many key enzymes and organisms are inactivated at high pH. The BEND compounds which release dye at lower pH are unsuitable for analytical determinations because their dye release is too slow.
Hence, there is a need in the art for a rapid and highly quantitative assay for analytes or microorganisms in aqueous liquids which can be carried out at physiological pH.